This application is a National Phase Application filed under 35 U.S.C xc2xa7371 of International Application No. PCT/JP97/02401, filed Jul. 10, 1997.
This invention relates to a novel use of MK to promote proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells in hematopoietic tissues, peripheral blood, or umbilical cord blood synergistically with other hematopoietic factors.
In blood, there exist various hemocytes having different shapes and functions, including erythrocytes, leukocytes, and platelets, which play important roles in maintaining homeostasis of the living body. These mature hemocytes have their own life-spans. For maintaining the hemocyte count at a constant level, hemocytes must be incessantly produced to make up for the number of hemocytes that is lost due to the expiration of their life-spans.
In the normal healthy individual, it is presumed that daily production of hemocytes reaches as much as 2xc3x971011 erythrocytes, 1011 leukocytes, and 1 to 2xc3x971011 platelets. Hematopoietic stem cells play central roles in the system to produce such an enormous number of hemocytes over a long period without being exhausted. The cells have not only self-renewal capability but also multipotentiality to differentiate to various mature hemocytes including erythrocytes, granulocytes, platelets, and lymphocytes. Hematopoietic stem cells (multipotential stem cells) lose their self-renewal capability as they proliferate to become hematopoietic precursor cells (committed stem cells) destined to differentiate to the specific hemocytes. Hematopoietic precursor cells then differentiate to mature peripheral hemocytes.
It has been known that a number of cytokines regulate each step of the hematopoietic system to proliferate and differentiate hematopoietic stem cells to various mature hemocytes via hematopoietic precursor cells. At least twenty kinds of these cytokines participating in the hematopoietic system have been found at present (Masami Bessho: Igaku no Ayumi 180(13): 802-806, 1997). The genes for all have been cloned, allowing their production on a large scale by genetic engineering techniques. Stem cell factor (SCF) and flk-2 ligand are the most remarkable cytokines as factors acting on mainly hematopoietic stem cells at the early stage of hematopoiesis. SCF acts on the most undifferentiated hematopoietic stem cells. In either mice or humans, it remarkably promotes the formation of colonies of blast colony-forming unit (CFU-BL), colony-forming unit-mixed (CFU-Mix), burst forming unit-erythrocyte (BFU-e), colony-forming unit-granulocyte/macrophage (CFU-GM), eosinophil colony-forming unit (CFU-Eo), and colony-forming unit-megakaryocyte (CFU-Meg), showing a synergistic effect with various cytokines such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-11, G-CSF, GM-CSF, and EPO. It has been reported that SCF alone has weak colony-stimulating activity (Tsuji, K. et al., Blood 78: 1223, 1991; Shioharu, M. et al., Blood 81: 1453, 1993; Kubo, T. and Nakahata, T., Int. Hematol. 58: 153, 1993). Nevertheless, SCF is thought to be the most important cytokine for in vitro amplification of hematopoietic stem cells at present.
The gene for flk-2 ligand has been just recently cloned and its biological activity has not been fully clarified. Since it exhibits synergistic actions with many cytokines as SCF does, it is expected to be an important factor for in vitro amplification of human hematopoietic stem cells.
Some of these hematopoietic factors have been clinically applied. For example, erythropoietin (EPO), which promotes the production of erythrocytes, is used for treating renal anemia, and granulocyte colony-stimulating factor (G-CSF), which promotes the production of neutrophils is used for treating neutropenia caused by cancer chemotherapy. These contribute to improved quality of life of patients. Recently, the clinical application of thrombopoietin (TPO) for treating thrombocytopenia has been studied because it promotes the production of platelets.
On the one hand, since hematopoietic stem cells are capable of reconstituting all kinds of cells in the hematopoietic system, the transplantation of hematopoietic stem cells has been widely performed for hematopoietic tumors. Recently, the transplantation of peripheral blood stem cells has rapidly become prevalent, and gathered attention as the powerful fundamental therapy for the chemotherapy-sensitive malignant tumors including the hematopoietic organ tumors. Furthermore, as a future prospect, the transplantation of hematopoietic stem cells is expected to be introduced to many cell therapy and gene therapy protocols. For that purpose, it is necessary to establish a method for amplifying hematopoietic stem cells in vitro. However, even now, human hematopoietic stem cells have been neither isolated nor clarified as to what extent they can repeat self-renewal.
An objective of the present invention is to provide a novel cytokine capable of synergistically promoting the proliferation and differentiation of hematopoietic stem cells or hematopoietic precursor cells in combination with known cytokines.
Another objective of the present invention is to provide a novel cytokine capable of promoting the proliferation and differentiation of granulocyte/monocyte precursor cells.
Still another objective of the present invention is to provide a novel cytokine capable of promoting the proliferation and differentiation of erythroblast precursor cells in combination with known cytokines.
The present inventors have found that single use of a novel growth factor called midkine (MK) or pleiotrophin (PTN) promotes the proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells (also called hematopoietic cells) of mammals such as mice and humans in vitro. MK also exerts an extremely remarkable synergistic effect for proliferating and differentiating hematopoietic cells when used together with SCF, M-CSF, G-CSF, GM-CSF, IL-3, and IL-6. Furthermore, the inventors have found that MK or PTN promotes rapid recovery of neutrophils in neutropenia of mammals.
The present invention will be described in detail below.
MK was isolated as the product of a gene that is expressed at the early stage of the differentiation of mouse embryonic tumor cells by the induction of retinoic acid (Kadomatsu, K., et al., Biochem. Biophy. Res. Commun. 115: 1312-1318, 1988). PTN was found as a heparin-binding protein with neurite outgrowth capability in the newborn rat brain (Rauvaa, H., EMBO J. 8: 2933-2941, 1989). MK and PTN belong to a new class of heparin-binding growth factors, sharing a 45% homology (in amino acid sequence) to each other, and called the MK family. MK and PTN respectively exhibit characteristic expression patterns in the developmental process, indicating that they have important physiological activities for the implementation of differentiation.
Paying attention to such biological activities of the MK family, the present inventors studied their hematopoietic factor activities to proliferate and differentiate myeloid cells and peripheral blood stem cells of mammals. In general, at what stage of the proliferation/differentiation process of hematopoietic stem cells and hematopoietic precursor cells in myeloid hematopoietic factors participate and function can be studied by culturing a certain number of myelocytes in a semi-solid medium in the presence of these hematopoietic factors, selecting cells constituting colonies formed, and counting the number of colonies. In such colony formation methods, it has been proved by a number of direct or indirect methods that, a single hematopoietic precursor cell proliferates, divides, and matures, forming a single colony comprising many matured hemocytes. There are colony formation assay methods specific for cells of each hematopoietic system including granulocyte/macrophage, erythroblasts, and megakaryocytes, and stimulators specific for each hematopoietic system are used.
Precursor cells of the granulocyte/macrophage system, CFU-GM, differentiate to precursor cells of neutrophil system, CFU-G, and precursor cells of monocytes, CFU-M. For that purpose, colony-simulating factors (CSF) specific to each precursor cell must be present. More specifically, GM-CSF is required for CFU-GM, G-CSF for CFU-G, and M-CSF for CFU-M. Some of these CSFs not only differentiate precursor cells to mature cells but also activate the function of matured hemocytes.
CFU-GM can be cultivated by either the soft agar method or the methylcellulose method using bone marrow nucleated cells. Since colonies formed by either method are constituted by a cell population of granulocytes and macrophages at various developmental stages, precursor cells one step further differentiated from hematopoietic stem cells are to be examined. Picking up and staining of these colonies revealed the presence of G colony consisting of granulocytes, M colony consisting of macrophages, and GM colony consisting of the mixture of both. In humans, colonies are rather small and classified into a group called a colony containing 40 cells or more and a group called a cluster with a lower accumulation of cells than a colony.
In order to examine whether MK has the activity to causing myelocytes to proliferate, the proliferation of mouse myelocytes was assayed by the MTT method, resulting in enhancing the proliferation 1.6- to 2-fold in the system supplemented with MK at the concentrations of 5, 50, 500, and 5000 ng/ml as compared with the system without MK. A concentration-dependent elevation of the activity was observed in the range of 5 to 500 ng/ml MK.
In the colony assay for human peripheral blood mononuclear cells in the presence of various cytokines, as shown in FIG. 1, colonies were not formed at all in the system without cytokines, but formed in the system supplemented with MK similarly as with GM-CSF and IL-3. The colony size tends to be larger in the system with MK added than in the systems with cytokines such as G-CSF, GM-CSF, and IL-3 added. These results indicate that MK alone has the activity to maintain the viability of human peripheral blood stem cells or hematopoietic precursor cells, or promote their proliferation. Furthermore, the combined use of MK with other cytokines such as M-CSF, G-CSF, GM-CSF, IL-3, and IL-6 synergistically promotes colony-forming capability. For example, the number of colonies increased 7 to 9-fold in the cases of combined use of MK with G-CSF, GM-CSF, or IL-3 as compared with those of the single use of MK, G-CSF, GM-CSF, or IL-3. Also, the combination of MK +GM-CSF+IL-3+IL-6 remarkably increased the number of colonies formed as compared with that of GM-CSF+IL-3+IL-6, and the combination of MK+G-CSF+IL-6 singnificantly increased the number of colonies as compared with that of G-CSF+IL-6. In another experiment using a source of human peripheral blood mononuclear cells different from that used for the experiments described in FIG. 1, treatment with MK, GM-CSF, or IL-3 alone produced primarily GM colonies, while use of G-CSF alone produced primarily G colonies. Combinations of MK+G-CSF+GM-CSF, or of MK+G-CSF significantly increased the number of G colonies as compared with the use of G-CSF alone. That is, MK is considered to synergistically promote the proliferation, differentiation and maturation of CFU-GM of G-CSF, increasing the number of neutrophils in the peripheral blood.
When cells after 2-week liquid culture of human peripheral blood stem cells in the presence of various cytokines were examined by specific staining, there were observed, as shown in FIG. 3, predominantly many granulocytes (neutrophils) in the system supplemented with MK, clearly indicating the action of MK on the proliferation of neutrophils. Especially, in the case the of combination of MK+G-CSF+GM-CSF+SCF +IL-3+IL-6, there was observed an extremely remarkable promotion of the proliferation and differentiation of neutrophils.
Also, in the colony assay performed after the above-described liquid culture, the cell adherence to a culture dish increased in the system supplemented with MK as compared with that without MK, indicating that MK also promotes the proliferation of the interstitial cell system (stroma cell system). In the case of IL-6 alone, colonies formed were of macrophages, and in the case of MK+IL-6, half of colonies formed were of granulocytes. These results indicate the participation of MK in the promotion of proliferation and differentiation of granulocytes.
Whether such a remarkable promotion by MK of the production of neutrophils is displayed in vivo can be studied by administering MK to a mouse whose hematopoietic system has been damaged by administration of an anticancer drug, or exposure to radiation, and examining the recovery state of neutrophils. MK was administered to a mouse daily for 13 consecutive days, and on the 5th day after the initiation of administration, an anticancer drug, Cyclophosphamide (CY), was administered to the mouse. Examination of hemocytes in the blood collected from the mouse at appropriate intervals revealed a remarkable promotion of the recovery of the number of neutrophils as expected (Table 1).
The action of MK on hematopoietic cells under the conditions closer to in vivo can be examined by, for example, the colony assay using a methylcellulose medium containing EPO, IL-3, IL-6, and SCF [MethoCult GF M3434 (Stem Cell Technologies, Inc.)]. This medium (hereinafter called M3434 medium) can proliferate and differentiate precursor cells of erythrocytes, leukocytes, and platelets.
Results of the colony assay for mouse spleen cells in the system of the M3434 medium supplemented with MK are shown in FIGS. 4 and 5. FIG. 4 illustrates the number of CFU-GM colony or CFU-G colony. Although CFU-GM colony or CFU-G colonies can be formed with the M3434 medium alone, the number of colonies generally increases in the system supplemented with the MK as compared with the M3434 medium alone. Especially, when MK is added at the concentration of 1 to 10 ng/ml, the colony number remarkably increased 2 to 3-fold on the 8th and 10th day of the assay. FIG. 5 illustrates the number of colony-forming unit-mixed (CFU-Mix). CFU-Mix are multipotential stem cells at a slightly differentiated stage, having lower self-renewal capability than blast colony-forming units (CFU-BL) which are the most undifferentiated cells identifiable by the in vitro colony assay and are thought to contain cells capable of differentiating to erythrocytes, leukocytes, and platelets. In the system supplemented with MK, CFU-Mix colony significantly increased in number. That is, MK, at least by its combined use with IL-3, IL-6, SCF and EPO, is thought to significantly promote the proliferation and differentiation of hematopoietic stem cells or immature hematopoietic cells close to them. These activities are thought to be very useful for the proliferation of hematopoietic stem cells in vitro for the transplantation of bone marrow and peripheral blood stem cells, or gene transfer to hematopoietic stem cells.
In the hematopoietic cells, the more mature peripheral blood cells are, the more sensitive to anticancer drugs. Utilizing this property, the present inventors attempted to concentrate hematopoietic stem cells or. hematopoietic precursor cells by an anticancer drug. Namely, the colony assay was performed using spleen cells of a mouse, to which Cyclophosphamide has been administered. Results are shown in FIGS. 6 and 7. FIG. 6 shows the number of CFU-Mix and CFU-G colonies increased 2-fold or more in the system supplemented with MK as compared with the system without MK. This experiment also indicates that MK promotes the proliferation of hematopoietic stem cells and hematopoietic precursor cells.
The effect of MK was investigated using peripheral hemocytes from a patient with non-Hodgkin""s lymphoma and a MethoCult H4230 medium [consisting of methylcellulose (0.9%), 2-mercaptoethanol (10 to 4 M), L-glutamine (2 mM), fetal bovine serum (30%), and bovine serum albumin (1%), and containing neither CSF nor EPO; Stem Cell Technologies Inc.]. The colony assay was performed using the following combinations; MethoCult H4230 alone, H4230+MK, H4230+G-CSF, and H4230+MK+G-CSF. The proportion of CD34 positive cells in peripheral stem cells from the patient was 1.4%. Results are shown in FIG. 8. Although no colonies were formed with MK alone, a remarkable colony-forming capability was manifested in the case of MK+G-CSF, and the number of colonies was clearly twice or more as high as that in the case of G-CSF alone. On and after the 10th day of the initiation of assay, the increase in number of colonies tends to reduce in the MK+G-CSF group as compared with the group of G-CSF alone. Microscopic observation of colonies on and after the 10th day revealed a tendency that the colony maturation was accelerated in the MK+G-CSF group as compared with the G-CSF alone group. Therefore, the deceleration of the increase in number of colonies in the MK+G-CSF group as compared with the G-CSF alone group is probably attributed to the accelerated maturation of colonies.
It is noteworthy that, in the above-described colony assay, the size of each of colonies formed was always larger in groups supplemented with MK as compared with groups with no MK added. In order to study this fact quantitatively, three each of large colonies formed on the 14th day in the presence of MK alone, G-CSF alone, and MK+G-CSF in the colony assay of peripheral blood stem cells from the above-described patient were selected, sucked up under a microscope, and counted for their constituting cells with a hemocytometer to calculate mean values. Results are shown in FIG. 9. It is obvious that colonies formed with MK+G-CSF contain more cells than those formed with G-CSF alone. That the size of colony is large means that the number of constituting cells is also large. From these results, it is evident that MK acts on the proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells.
The colony assay was similarly performed with the peripheral blood from a healthy normal individual. The proportion of CD34-positive cells in the peripheral blood of this subject was 0.4%. Results are shown in FIG. 10. Colonies were formed with MK alone. On the 10th and 14th days, the number of colonies increased MK concentration-dependently. The MK+G-CSF system produced at the highest 2 or more times as many colonies as the system of G-CSF alone. These results clearly shows that, when MK was added, the same tendency was obtained regardless of whether cells are derived from a healthy normal individual or a patient.
Furthermore, the colony assay was similarly performed using the peripheral blood from the above-described healthy normal individual in the presence of pleiotrophin (PTN), which is another member of the MK family. PTN used herein was a recombinant PTN [pleiotrophin, recombinant human (Sf21-derived); Lot GH055011) (R and D Systems)]. Results are shown in FIG. 11. PTN alone exhibited the colony-forming capability of MK and the number of colonies formed was markedly high. A synergistic action of PTN with G-CSF to promote the colony formation was similarly observed as in the case of MK. From these results, PTN obviously promotes the proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells like MK.
Next, using a hematopoietic stem cell assay medium, complete type (Lot No. 96101601; Kyokuto Seiyaku Kogyo) containing IL-3, SCF, G-CSF, and EPO, the colony assay was carried out with peripheral blood cells from a healthy normal individual. This assay is considered to be performed under conditions closer to in vivo. Results with MK are shown in FIGS. 12 and 13, and those with PTN in FIGS. 14 and 15. No increase in BFU-E was observed with any combinations including MK+IL-3, MK+SCF, and MK+G-CSF. However, the combinations of MK+EPO and PTN+EPO were assumed to increase BFU-E. Erythroblast precursor cells, BFU-E, were formed on the 14th day of culture, and are known to be more undifferentiated than CFU-E formed on the 5th to 7th days. Addition of MK or PTN to a Kyokuto complete medium resulted in formation of at the highest 2 or more times as many BFU-E as the complete medium alone on the 12th day after the initiation of culture. These results indicate that at least the addition of MK to the complete medium results in promoting the proliferation of erythroblasts as well. As described above, it is evident that the MK family is capable of acting on hematopoietic stem cells and hematopoietic precursor cells in the hematopoietic tissues of mammals to maintain, proliferate, and differentiate them, and synergistically or additionally enhancing the above-described functions by the combined use with various cytokines such as SCF, M-CSF, G-CSF, GM-CSF, IL-3 and IL-6. Especially, the MK family remarkably promotes the proliferation of CFU-Mix, which is very close to multipotential stem cells, under conditions closer to in vivo. The MK family also promotes the proliferation and differentiation of granulocyte/macrophage precursor cells and exerts the remarkable neutrophil increasing effect in an in vivo neutropenia model. This MK family alone or in combination with more than one cytokine including SCF, M-CSF, G-CSF, GM-CSF, IL-3, and IL-6 can be clinically applied and, especially, used for the ex vivo expansion of hematopoietic stem cells in the transplantation of bone marrow and stem cells derived from the peripheral blood and umbilical cord blood. In addition, the MK family is expected to be used for the treatment patients with and prevention of neutropenia, vertebrate anemia, and leukemia caused by cancer chemotherapy. Furthermore, the MK family would be used in the future for proliferating stem cells for gene therapy targeting hematopoietic stem cells. Especially, it is very promising to increase the dose density in cancer chemotherapy by the combined use of MK with G-CSF, improving effects of chemotherapy by increasing the dose of antitumor drugs or shortening the administration period.
MK and PTN used in the present invention can be either a natural or recombinant product. The recombinant MK family means a substance well homologous with the natural MK or PTN and having biological activities equivalent thereto. Such MK or PTN includes their derivatives and analogues. The purified MK or PTN of mammals means those derived from mice or humans, but not limited thereto. MK or PTN of this invention also includes glycosylated and non-glycosylated MK or PTN.